Natural killer (NK) function is mediated by cell membrane receptors belonging to several different gene families that detect a variety of ligands or absence thereof on the surfaces of various target cells. The full complement of receptors that mediate NK function is likely not understood. We have cloned the novel immune-type receptor (NITR) genes, which constitute a large multigene family encoding transmembrane immunoglobulin (Ig)-type molecules. NITRs exhibit structural characteristics of NK receptors (including potential activating/inhibitory transmembrane signaling) but possess ectodomains of the Ig/T cell antigen receptor (TCR) variable region type. NITR genes, which have been identified in zebrafish (a significant developmental and genetic model system), do not undergo somatic reorganization, and represent the only example of multigenic receptors outside of Ig and TCR genes in which diversified variable regions are likely utilized as the core recognition element of a receptor family. NITRs occupy a unique and potentially highly informative link between the innate and adaptive immune systems. A genomics-based approach, which is being coordinated through the Zebrafish Genome Project, will create a transcriptional map of the NITR gene cluster, identify the central features of this genetic region, and define the full range of NITR-related genes in this species. Genomics studies conducted to date have identified seven families of NITRs in zebrafish, of which six possess cytoplasmic ITIMs and are designated as inhibitory type. Members of a seventh family (nitr5) encode a neutral transmembrane region and an ITAM-containing cytoplasmic tail. Based on our preliminary findings that inhibitory NITRs expressed in human NK cells can transduce negative regulatory signals, epitope-tagged NITR constructs will be transfected into human NK cells in order to define their surface biochemistry, identify potential associations with putative adaptor proteins and further characterize the unique direct activating receptors of the nitr5 family. A major effort will be directed to the identification of NITR ligands employing soluble bivalent NITR-IgGFc and pentavalent NITR-IgMFc chimeras as well as an NITR ectodomain-CD3zeta-NFAT-coupled GFP reporter assay. Both direct binding and reporter signaling assays will be used to examine interactions of NITRs with candidate ligands, including chimeric zebrafish-human MHC I, which will be expressed in human cells as well as chimeric zebrafish-medaka MHC I, which will be expressed in transgenic Oryzias latipes (medaka; an unrelated bony fish). Non-MHC I ligands will be expressed in an adenovirus system and several different recombinant library-screening approaches, as well as potential direct binding to pathogens will be employed to identify potential endogenous and exogenous ligands. Collectively, this multifaceted approach will define the essential structure/function relationships of a novel family of putative recognition molecules in an informative vertebrate developmental model system. Such information is significant to understanding the evolution and interrelatedness of NK and other immune recognition processes.